The free, or non-bound, fraction of many drugs and hormones in blood or serum is of great interest to clinical and pharmaceutical chemists since this is believed to represent the active form of such agents. This makes this form ideal as an analytical tool for patient diagnosis and treatment. However, there is currently no general and fast approach for measuring the free fractions of drugs and hormones in clinical samples. The overall goals of this proposal are 1) to obtain a better understanding of drug and hormone interactions with their binding agents in blood or serum and 2) to use this information to develop fast, reliable free drug and hormone assays. The underlying hypothesis of this project is that a new class of improved free drug and hormone assays can be created based on the techniques of ultrafast affinity extraction and chromatographic immunoassays, as recently demonstrated for compounds such as warfarin, thyroxine, phenytoin and carbamazepine. Future studies will build on these efforts by considering new analytes and improvements in the methods used for such work. Advances to be explored will include the development of affinity columns for studying the binding of drugs to serum lipoproteins; the creation of microaffinity columns for rapid studies of drug/hormone binding and/or the entrapment of their binding agents; and the use of new methods for solution-phase kinetic studies of drug and hormone binding to serum agents. Improved tools to be created for the measurement of free drug and hormone fractions will include restricted access affinity supports; serum protein columns for ultrafast affinity extractions; multidimensional methods for free fraction measurements; and the reverse displacement immunoassay as an alternative to ultrafast affinity extraction. This work will provide a better understanding of how drugs and hormones interact with serum agents and result in faster, more convenient techniques for free drug and hormone measurements. This, in turn, will provide clinicians with improved methods for studying how such compounds behave and interact in the body. [unreadable] [unreadable]